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Document heading doi: ©2015 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

Influence of Spirulina platensis exudates on the endocrine and nervous systems of a mammalian model

Samah Mamdouh Mohamed Fathy1,2, Ashraf Mohamed Mohamed Essa2,3* 1Zoology Department, Faculty of Science, Fayoum University, Fayoum, Egypt2Biology Department, Faculty of Science, Jazan University, Jazan, Kingdom of Saudi Arabia3Botany Department, Faculty of Science, Fayoum University, Fayoum, Egypt

Asian Pac J Trop Biomed 2015; 5(6):

Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Contents lists available at ScienceDirect

*Corresponding author: Ashraf Mohamed Mohamed Essa, Botany Department, Faculty of Science, Fayoum University, Fayoum, Egypt; Biology Department, Faculty of Science, Jazan University, Kingdom of Saudi Arabia. Tel: +9660566511430 E-mail: ashraf.essa@yahoo.com

1. Introduction

Spirulina is a filamentous, spiral-shaped, multicellular and

photosynthetic cyanobacteria. It is dominating the microflora

of alkaline saline waters with pH up to 11.0 and can be found

in diverse kinds of environments such as soils, fresh, brackish,

seawaters as well as industrial and domestic wastewater. Spirulina

contains up to 70% complete protein that contains all the essential

amino acids[1]. It is also rich in essential fatty acids, vitamins,

minerals, photosynthetic pigments, polysaccharides, glycolipids and

sulfolipids[2]. This cyanobacterium is cultivated around the world

and is used as a primary human dietary supplement[3,4]. It has been

stated by National Aeronautics and Space Administration (NASA)

that the nutritional value of one kilogram of Spirulina is equivalent

to one thousand kilogram of fruits and vegetables. Therefore in

long-term space missions, NASA proposed that Spirulina served

as a major source of food and nutrition[5]. It is also used as a feed

supplement in the aquaculture and poultry industries[6,7].

In addition to the nutritional advantages, Spirulina has other

ARTICLE INFO ABSTRACT

Objective: To investigate the effect of intra-peritoneal injection of purified exudates of axenic Spirulina platensis on the mammalian endocrine and nervous systems. Methods: The intra-peritoneal injection of the cyanobacterial exudates in mice was applied. Sex hormonal levels of testosterone and progesterone were measured using radioimmunoassay while the follicular stimulating hormone and luteinizing hormone were evaluated by direct chemiluminescence. In addition, superoxide dismutase, catalase and glutathione peroxidase were monitored in the hippocampus region using spectrophotometric method. The levels of the hippocampal monoamines, dopamine, noradrenaline and serotonin were analyzed by high performance liquid chromatography while the acetyl choline neurotransmitter was measured by colorimetric method using choline/acetylcholine assay kit. Results: A sharp disruption in the sex hormones levels of testosterone, progesterone, follicular stimulating hormone and luteinizing hormone was demonstrated in the serum of the treated mice. At the same time, a significant reduction in the endogenous antioxidant defense enzymes, superoxide dismutase, catalase and glutathione peroxidase was observed in the hippocampus region of the injected mice. Moreover, levels of dopamine, noradrenaline, serotonin and acetyl choline neurotransmitter in the same region were significantly affected as a result of the treatment with Spirulina filtrate. The gas chromatography-mass spectrometer and liquid chromatography mass spectrometry/mass spectrometry analysis showed the presence of some sterol-like compounds in the cyanobacterial filtrate. Conclusions: This study demonstrated the capability of Spirulina to release detrimental bioactive metabolites into their surrounding that can disrupt the mammalian endocrine and nervous systems.

Article history:Received 2 Feb 2015Received in revised form 10 Feb 2015Accepted 18 Mar 2015Available online 9 Apr 2015

Keywords:

Spirulina platensisSterol-like compoundsSex hormonesAntioxidant enzymesNeurotransmitters

Samah Mamdouh Mohamed Fathy and Ashraf Mohamed Mohamed Essa/Asian Pac J Trop Biomed 2015; 5(6): 931

beneficial characteristics such as antibacterial, antifungal, antiviral,

molluscicidal, anticancer, anti-inflammatory and antioxidant

activities as well as its stimulant effects on innate and specific

immunity[8-13]. Moreover, Spirulina was used successfully for the

clinical improvement of anaemia in children and the protection

against hay fever[14,15].

On the other hand, minor contraindications of using Spirulina

as a human dietary supplement including headache, muscle

pain, sweating, difficulty concentrating, and skin reactions

were recorded[16]. At the same time, Spirulina may contain the

hepatotoxin, microcystin, which is accumulated in the liver and can

potentially cause cancer or other liver diseases[17]. Furthermore,

Jiang et al. reported the existence of microcystins in 36 kinds of

cyanobacteria Spirulina health food samples obtained from various

retail outlets in China[18]. About 94% of the samples contained low

concentrations of microcystins. They concluded that the possible

potential health risks from chronic exposure to these toxins should

not be ignored, even if the toxin concentration was low.

Another harmful effect of Spirulina platensis (S. platensis) was

recorded by Mazokopakis et al. who correlated the ingestion of

Arthrospira platensis with a human life threaten disease called

rhabdomyolysis[19]. This disease leads to release of muscle

cell contents into the systemic circulation. Symptoms of acute

rhabdomyolysis including muscular weakness, swelling, pain,

cramping and tea-colored urine were recorded with a patient taking

Arthrospira platensis as a dietary supplement.

It has been reported that certain cyanobacterial species can produce

the neurotoxic amino acid β-N-methylamino-L-alanine (BMAA)[20,21]. The exposure to such these metabolites might be involved in

the etiology of certain neurodegenerative diseases[22]. Subsequent

testing of a variety of cyanobacterial species revealed that over 90%

of the tested genera could produce BMAA[21,23]. Additionally, it has

been recorded that under certain conditions, Spirulina, seems to be

able to produce some metabolites that can induce a neurological

disease (amyotrophic lateral sclerosis-parkinsonism-dementia

complex)[24].

In our previous study[25], it was shown that the intra-peritoneal

injection of some cyanobacterial exudates in mice demonstrated

a remarkable disruption of serum sex hormonal levels. Hence, the

aim of this study is to investigate the effect of the intra-peritoneal

injection of S. platensis exudates on the serum sex hormones as

well as the antioxidant enzymes and the neurotransmitters in the

hippocampus region of the treated mice.

2. Materials and methods

2.1. Ethics statement

This study was carried out in strict compliance with the Guidelines

of Animals Health Research Institute, Egypt. The study protocol

was approved by the Committee on the Ethics of Animals Health

Research Institute, Egypt (Permit Number 362 approved on August

31, 2010). Mice were sacrificed by decapitation after narcotizing

with aerosolized isoflurane. Regarding water samples collection

from lakes of Wadi El-Natrun, Egypt, there were no specific

permissions required and the field studies did not involve endangered

or protected species.

2.2. Isolation and growth of S. platensis

Water samples were collected in 2010 from lakes of Wadi El-

Natrun, Egypt (no specific permissions were required). Samples

were filtered through sterilized Whatman No. 41 filter paper and

then suspended on 5 mL sterilized Zarrouk medium[26]. One to two

drops of each suspension were inoculated on solid Zarrouk medium

and incubated for about two weeks in culture room at (25 ± 1) °C

under controlled continuous illumination of 40 µEm-2s-1. The plates

were examined and the best colonies were selected, picked up and

re-streaked to new agar plates. Restreaking and subculturing were

repeated several times to obtain unialgal cultures. To get axenic

culture, the tested cyanobacterium was grown in liquid cultures for

12 d to attain vigorous growth. About 20 mL of each culture was

centrifuged at 6 000 r/min for 10 min and the algal pellets were then

streaked on peptone or yeast extract solid medium. Those which

proved not to be axenic streaks have been repeated till they become

axenic. Inocula from axenic cultures were taken into sterilized

liquid medium to be ready for the desired experiments. The purified

cyanobacterium was identified as S. platensis[27].

Batch culture of S. platensis was grown on Zarrouk medium for

28 d under the previous conditions and cells were separated from

their culture by filtration using Whatman No. 41 filter paper. The

cyanobacterial biomass was subjected to chlorophyll “a” analysis

while the culture filtrate was stored at 4°C. Chlorophyll “a” content

in the biomass was extracted using the standard acetone extraction

method[28]. After extraction, chlorophyll “a” was determined

spectrophotometrically at 750 and 665 nm using a PYE Nnicom, SP8-

100 UV spectrophotometer.

2.3. Purification of S. platensis exudates

The culture filtrate from the previous step was passed through glass

fiber filters and 0.45 mm Millipore membranes. The filtrate was then

concentrated in a rotary evaporator at 40 °C under reduced pressure,

dialyzed successively against running tap water and distilled water

in a spectra/por dialysis tube (Spectra/Por; Spectrum Medical

Industries, Los Angeles, CA) with a molecular weight cut-off of

3 500 daltons, and freeze dried. Weighed portions of lyophilized

staff were suspended in deionized water and dispersed by storage for

18 h at 4 °C. After that, the solution was purified using C18 solid-

phase extraction discs (Empore, 3M, Minneapolis, MN, USA). Prior

to extraction, the discs were conditioned with methanol and water.

The Spirulina filtrate was extracted with 47 mm discs by applying

vacuum to maintain a flow rate of 0.5 to 5 mL/min. The C18 discs

were eluted three times with 5 mL of methanol each time which was

blown down to dryness and stored frozen. The content was dissolved

in physiological saline solution (1 mg/mL), sterilized by filtration

through a 0.22 µm filter and used for intra-peritoneal injection of

the mice. Another set of the purified exudates was dissolved in

Samah Mamdouh Mohamed Fathy and Ashraf Mohamed Mohamed Essa/Asian Pac J Trop Biomed 2015; 5(6): 932

methanol/water (65/35, v/v) and used for the gas chromatography-

mass spectrometer (GC-MS) and liquid chromatography–mass

spectrometry (LC-MS)/MS analysis.

2.4. Experimental animals

Fifty male albino mice weighing 20-25 g were housed in groups

of five in polyethylene cages in a temperature controlled room

under 12/12 h light/dark cycle and had free access to food pellets

and tap water ad libitum. The minimal number of animals needed

to obtain statistical significance was used and all efforts were made

to minimize animal suffering. On the day of experiment, mice

were divided into two groups, one group received 5 intra-peritoneal

injections of 15 mg/kg of the purified S. platensis exudates in saline

at 24 h intervals (total dose: 75 mg/kg) while the control set was

given equal dose of physiological saline solution (0.85% NaCl).

2.5. Blood collection and sample preparation

One day post injection, mice were briefly anesthetized with

aerosolized isoflurane and decapitated. The collected blood samples

were placed at room temperature for approximately 30 min. Then,

the tubes were centrifuged at 3 000 g for 10 min and the supernatant

was collected for the assaying of sex hormones. Fresh brain samples

were obtained and partitioned using a dissecting microscope.

Hippocampal samples were weighed and homogenized to give 10%

(w/v) homogenate in 100 mL of ice-cold dissolution buffer [0.1

mol/L perchloric acid, 0.1 mmol/L sodium bisulfite, and 0.1 mmol/

L ethylene diamine tetraacetic acid (EDTA)]. All homogenates were

centrifuged at 20 000 g for 25 min at 4 °C. Supernatants were filtered

through 0.22 mm pore size polyvinylidene fluoride membrane filters

(Millipore Corp., Bedford, MA, USA) and immediately frozen and

stored at -80 °C for the analysis of the monoamines.

Another set of the hippocampal tissues were weighed and

homogenized to give 10% (w/v) homogenate in ice cold medium

containing 50 mmol/L Tris-HCl and 300 mmol/L sucrose at pH 7.4.

The homogenate was centrifuged at 20 000 g for 25 min at 4 °C. The

supernatant was filtered through polyvinylidene fluoride membrane

filters and stored at -80 °C for the analysis of the antioxidant

enzymes.

2.6. Assay of sex hormones

Serum concentrations of total testosterone, free testosterone

and progesterone were measured by radioimmunoassay using a

commercial kit obtained from BioSource (Nivelles, Belgium). In

addition, luteinizing hormone and follicle stimulating hormone

(FSH) were measured by direct chemiluminescence (ADVIA Centaur,

Bayer Co., Bayer AG, Leverkusen, Germany).

2.7. High-pressure liquid chromatography (HPLC) with electrochemical detection analysis of the monoamines

The monoamines of hippocampal homogenates were analyzed

using HPLC with electrochemical detection. The analysis was

performed with an ESA Model 5600A Coul Array® system (ESA Inc.,

Chemlsford, MA, USA), equipped with Shimadzu Model DGU-14A

on-line degassing unit (Shimadzu Scientific Instruments, Columbia,

MD, USA), an ESA Model 582 pump and an ESA Model 542

refrigerated autosampler. Electrode potentials were selected over the

range of 0 to +700 mV, with a 50 mV increment against palladium

electrodes. Chromatographic separation was achieved by auto-

injecting 30 µL sample aliquots at 5 °C onto a MetaChem Intersil

(MetaChem Technologies, Torrance, CA, USA) reversed-phase C18

column with an ESA Hypersil C18 precolumn. A mobile phase flow

rate of 1.25 mL/min and analysis time of 45 min were used for all

experiments. System control and data processing were performed

using ESA CoulArray software (version 1.02). The mobile phase

containing 75 mmol/L monobasic sodium dihydrogen phosphate, 2

mmol/L sodium octylsulphonate, 25 mmol/L EDTA and 100 mL of

triethylamine was prepared in 1 800 mL of HPLC grade water. This

solution was then mixed with 200 mL of HPLC grade acetonitrile

and buffered to pH 3.0 using concentrated ortho-phosphoric acid.

Then, the mobile phase was filtered through a 0.20 mm pore size

white nylon filter membrane (Millipore Corp.) and degassed under

vacuum for 30 min prior to use. Stock standard solutions were

prepared by dissolving 10 mg of dopamine (DA), serotonin (5-HT)

and noradrenaline (NA) in 25 mL of dissolution buffer. These

concentrates were then divided into 1 mL aliquots, frozen, stored at

-80 °C and thawed prior to use at 4 °C. All samples were processed

in technical triplicate with median values used for mean calculations.

2.8. Determination of antioxidant enzymes activity

The hippocampal supernatant was used for the biochemical

analysis of the antioxidant enzymes. Superoxide dismutase

(SOD) activity was determined using Biodiagnostic Kit No. SD

25 21 (Biodiagnostic Co., Cairo, Egypt) which is based on the

spectrophotometric method[29]. This assay relies on the ability of the

enzyme to inhibit the phenazine methosulphate-mediated reduction

of nitro blue tetrazolium dye and the absorbance was measured at 560

nm. Catalase (CAT) activity was measured using Biodiagnostic Kit

No. CA 25 17 (Biodiagnostic Co., Cairo, Egypt) which is based on

the spectrophotometric method[30]. CAT reacts with a known quantity

of hydrogen peroxide and the reaction is stopped after 1 min with

CAT inhibitor. In the presence of peroxidase, the remaining hydrogen

peroxide reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid

and 4-aminophenazone to form a chromophore with a color intensity

inversely proportional to the amount of CAT in the sample. The

absorbance was measured at 510 nm. Glutathione peroxidase (GPx)

activity, which catalyzes the oxidation of reduced glutathione to

glutathione disulfide, was assayed spectrophotometrically[31]. The

reaction mixture consisted of 240 mIU/mL of glutathione disulfide

reductase, 1 mmol/L glutathione, 0.15 mmol/L nicotinamide adenine

dinucleotide phosphate in 0.1 mol/L potassium phosphate buffer, pH

7.0, containing 1 mmol/L EDTA and 1 mmol/L sodium azide; a 50 µL

sample was added to this mixture and allowed to equilibrate at 37 °C

for 3 min and the absorbance was measured at 340 nm.

Samah Mamdouh Mohamed Fathy and Ashraf Mohamed Mohamed Essa/Asian Pac J Trop Biomed 2015; 5(6): 933

2.9. Determination of acetylcholine (Ach)

Ach level was measured by colorimetric method using choline/Ach

assay kit purchased from Biovision Research Products Co., Linda

Vista Avenue, Mountain View, CA, USA[32].

2.10. Analysis of sterol-like compounds 2.10.1. GC-MS analysis GC-MS analysis was carried out in Faculty of Science (Fayoum

University) on an Agilent 5973 single quadrupole mass spectrometer

(Palo Alto, CA, USA), coupled with an Agilent 6890 gas

chromatograph used with an Agilent DB-5.625 (30 mm × 0.25 mm inner diameter × 0.25 µm) analytical column (inlet temperature:

275 °C; injection volume: 2 µL; J&W Scientific, Folsom, CA, USA).

The total GC run time was 53 min, and the carrier gas was helium.

The initial oven temperature was held at 80 °C for 2 min and then

increased by 10 °C/min till it reached 290 °C, after which it was

held at this temperature for 30 min. The injector temperature was

290 °C and the split ratio was 1:30. National Institute of Standards

and Technology mass spectral library was used to identify the

compounds in the extracts using hexane as a control. The closest

match with the highest probability in the library was recorded.

2.10.2. LC tandem MS analysis The used MS system was a Quattro Micro mass spectrometer

(Waters, Milford, MA, USA). High purity nitrogen was used as the

drying and electrospray ionization (ESI) nebulizing gas and was set

at 100 and 500 L/h, respectively. Argon was used as the collision gas

with a gas cell pirani pressure of 3.87e-3 mbar. The capillary voltage

was set at 3.5 kV and the source block and desolvation temperatures

were 100 and 300 °C, respectively. Chromatography was performed

using a Waters Alliance 2695 (Waters, Milford, MA). The column

was a Water Symmetry® C18, 2.1 mm × 50 mm, 3.5 um particle

size, with a guard column. The flow rate was 0.3 mL/min and an

injection volume of 10 uL was used. The run time of the electrospray

positive ionization (ESI+) mode was 60 min and the mobile phase

consisted of methanol/water (65/35, v/v) containing 0.3% formic

acid.

2.11. Statistical methods

Results are presented as mean ± standard error of the mean. Data

were compared for significant differences using Statistica Program

(version 5) and student’s t-test. The levels of significance chosen

were P < 0.05 and P < 0.01.

3. Results

3.1. Effect of S. platensis exudates on the level of sex hormones

Data shown in Table 1 clarified a sharp change in the serum level

of sex hormones of the male mice that were intra-peritoneally

injected with the Spirulina exudates. A massive reduction in the total,

free testosterone and progesterone was recorded (75.7%, 72.9% and

32.9%, respectively) compared with the untreated mice. On the other

hand, the levels of FSH and LH were significantly elevated in the

treated mice (30.9%, 40.1%). Table 1

Levels of testosterone, progesterone, FSH and luteinizing hormone in the

serum of male mice that were intra-peritoneally injected with the exudates

of S. platensis compared with the control treated with saline solution.

Sex hormones Saline solution 0.85 g/L NaCl

Exudates of S. platensis

Total testosterone 48.34 ± 2.76 11.76 ± 1.43Free testosterone 11.05 ± 1.56 2.99 ± 1.08Progesterone 1.91 ± 0.39 1.28 ± 0.41FSH 3.98 ± 0.48 5.21 ± 0.94LH 1.05 ± 0.26 1.47 ± 0.32

3.2. Effect of S. platensis exudates on the hippocampal activity of antioxidant enzymes

Data in Table 2 indicated that the injection of Spirulina exudates

led to a sharp decrease in SOD activity of the hippocampus by

42.9%. Similarly, reduced activities of CAT (8.5%) and GPx (24.9%)

were detected in the hippocampus of the treated mice compared to

the untreated mice.

Table 2

Effect of S. platensis exudates on the activity of the endogenous antioxidant

enzymes in the hippocampal region of the treated mice.

Treatment Endogenous antioxidant enzymes (IU/10 mg)

SOD CAT GPx

Saline solution (0.85 g/L) 10.66 ± 1.48 33.25 ± 2.86 8.15 ± 0.91

Exudates of S. platensis 6.08 ± 0.97 30.41 ± 1.83 6.12 ± 1.02

3.3. Effect of S. platensis exudates on the hippocampal level of neurotransmitters

The collected data (Table 3) demonstrated that the injection of

mice with Spirulina exudates induced variable changes in the level

of monoamine neurotransmitters in the hippocampus. A significant

decline in 5-HT (21.7%) was obtained, while DA and NA were

increased by 16.4% and 9.5% respectively. Meanwhile, a significant

reduction of the Ach concentration was recorded in the hippocampus

of the treated group (25.1%). Table 3

Effect of S. platensis exudates on the level of the neurotransmitters in the

hippocampal region of the treated mice.

Treatment Neurotransmitters (ng/10 mg)

DA NA 5-HT Ach

Saline solution (0.85 g/L) 31.25 ± 2.17 0.63 ± 0.42 151.05 ± 3.91 5.46 ± 1.73

Exudates of S. platensis 36.37 ± 1.95 0.69 ± 0.35 118.34 ± 2.94 4.09 ± 0.68

3.4. Identification of sterol-like hormones

The GC-MS analysis of the purified filtrates of S. platensis showed

the presence of sterol-like compounds (Table 4). The identity of

most peaks was determined by direct comparison to National

Institute of Standards and Technology GC-MS chemical library.

Compounds with retention times (17.13 min, 16.32 min, 15.95 min

and 13.67 min) showed the highest similarity with corticosterone

(52.1%), 2-methoxyestrone (69.8%), hydrocortisone (74.6%)

Samah Mamdouh Mohamed Fathy and Ashraf Mohamed Mohamed Essa/Asian Pac J Trop Biomed 2015; 5(6): 934

and androsterone (55.5%), respectively. Due to the low similarity

percentage between steroidal compounds of the S. platensis exudates

and the available eukaryotic steroids, LC-MS/MS was used with

cholesterol as reference[25]. Cholesterol molecule was broken down

into product ions (88.30, 106.22 and 256.34 g/mol); the later was

considered as a nucleus for the sterol-like molecules. Consequently,

certain sterol-like molecules (Figure 1) with molecular mass (328.1,

354.7, 504.3 and 859.8 g/mol) were identified in the exudates of S.

platensis at retention time 1.09 min, 2.08 min, 2.32 min and 2.78

min, respectively.

Table 4

Bioactive compounds identified from the culture filtrate of S. platensis

revealed by GC-MS.

Retention time (min)

Molar mass (g/mol)

Close match Similarity (%)

17.13 358 Corticosterone 52.116.32 356 2-methoxyestrone 69.815.95 316 Hydrocortisone 74.613.67 302 Androsterone 55.5

Data are representative of at least three independent biological replicates.

2524232221201918171615141312111098

Rel

ativ

e ab

unda

nce

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

RT: 0.30

RT: 1.09

RT: 2.78

RT: 1.56

RT: 2.08

0.56

2.32

2.57

0.80

Figure 1. LC-MS/MS chromatograms for the analysis of the exudates of S.

platensis.

Black arrows indicate the beaks that contain the sterol-like nucleus

(256.32).

S. platensis

Sterol-like compounds

Sex hormones disruption

Reduction of antioxidant enzymes activity in

hippocampus

Reduction of Ach level in hippocampus

Fluctuation of monoamines in hippocampus

Induction of hippocampal neurodegeneration

Apparent neurological symptoms such as ataxia, convulsion and comma

Figure 2. Schematic representation summarizing the effect of sterol-like

compounds of S. platensis on level of sex hormones, antioxidant enzymes

activity, monoamine neurotransmitters and Ach concentration in the

hippocampal region of the treated mice.

4. Discussion

Although Spirulina possessed various positive nutritional and

therapeutic properties, few studies reported its side effects. The

current study clarified a sharp disturbance in the level of the sex

hormones, testosterone, progesterone, FSH and LH of male mice

that were intra-peritoneally injected with the Spirulina exudates.

The fluctuation in the sex hormonal concentrations was attributed

to the presence of certain bioactive compounds in the culture

filtrate. These compounds might have a dysregulative effect on the

reproductive hormones. In our previous study, it was shown that

the injection of mice with the exudates of certain cyanobacterial

species (Nostoc ellipsosporum, Nostoc muscorum, Anabaena oryzae

and Anabaena sp.) led to a marked disruption in the serum level of

the reproductive hormones[25]. At the same time, Ding et al. and

Damkova et al. demonstrated that the exposure of vertebral models

to the cyanobacterial extract induced negative consequences on the

reproductive system such as sex hormones in addition to damaged

testes and low quality of mature sperm[33,34].

The GC-MS and LC-MS/MS analysis showed the presence of sterol-

like compounds in the exudates of Spirulina. In accordance with that,

some studies clarified the existence of steroidal compounds such as

cholesterol, beta-sitosterol, campesterol and stigmasterol in the dry

mass of Spirulina[35,36]. Moreover, Ramadan et al. identified the

lipid profile of S. platensis and clarified the existence of esterified

sterols and certain free sterols in their extract[37].

The presence of sterol-like compounds in the culture filtrate of

Spirulina might be responsible for the disruption of sex hormones

via interfering with the hormone-dependent signaling pathways.

Sterol-like compounds can increase or block the metabolism of

naturally occurring steroid hormones via activating or antagonizing

nuclear hormone receptors[38]. Moreover, these compounds may

cause perturbations in the level of gonadotropin-releasing hormone

which in turn affect the synthesis and the release of FSH and LH[39].

Regarding the level of neurotransmitters in the hippocampus

region, the obtained results verified that both DA and NA levels

were slightly elevated while a significant decline in 5-HT and Ach

levels was observed in mice treated with Spirulina exudates. The

fluctuation of neurotransmitters levels was correlated with the

change in the level of sex hormones in the treated mice due to the

effect of the sterol-like compounds of the culture filtrate. These

results are in agreement with Cornil et al. who demonstrated that

the injection of estradiol rapidly activated male sexual behavior

in quail[40]. This effect was associated with changes in the

concentration of monoamines and their activity. Additionally, Tucci

et al. studied the effect of stanozolol, a synthetic anabolic steroid

derived from dihydrotestosterone, on the level of monoamines in rat

brain[41]. The treated rats showed neurochemical modifications of

DA in hippocampus and prefrontal cortex while 5-HT was changed

in all brain areas. In addition, the decline of Ach in response to

sex hormonal alteration was in agreement with Takase et al. who

supported the correlation between sex hormonal change and Ach

level preservation in hippocampus[42].

The treated mice exhibited neurological symptoms comprising

Samah Mamdouh Mohamed Fathy and Ashraf Mohamed Mohamed Essa/Asian Pac J Trop Biomed 2015; 5(6): 935

ataxia, convulsion and comma that were recovered one hour after

each injection. These signs might be attributed to perturbation

in monoamine neurotransmitters. It has been reported that

change in monoamines was observed in various neurological and

psychiatric disorders such as addiction, depression and attention

deficit/hyperactivity disorders[43]. Consequently, the disruption

of sex hormones level could exert direct or indirect effect on

the mammalian central nervous system either via regulating the

expression of specific hormone-sensitive genes or by modulating

the activity of neurotransmitter receptors[44,45].

The current study indicated a remarkable decrease in the

level of the antioxidant enzymes (SOD, CAT and GPx) in the

hippocampus of mice treated with Spirulina exudates. Under

normal physiological conditions, there is a critical balance in the

generation of oxygen free radicals and antioxidant defense system

used by organisms to deactivate and protect themselves against

free radical toxicity. As a result of the interference of the steroidal

compounds identified in the Spirulina filtrate with sex hormone

receptors, a marked disruption in the level of the sex hormones was

observed. The alterations of the steroidal hormones concentration

might be responsible for the reduction of the antioxidant enzymes

activity. This hypothesis is consistent with Pajovic and Saicic

who showed that the endogenous patterns of antioxidant defense

enzyme expression are modulated by sexual steroid hormones[46].

Furthermore, Pajovic et al. and Michos et al. demonstrated that

the enzyme activity of the antioxidant defense system in the brain

tissue of female and male rats indicated a certain dependence on

the concentration of steroidal hormones such as progesterone

and estrogen[47,48]. Moreover, the decline in the abovementioned

antioxidant enzymes along with Ach level as a response for the

injection of Spirulina exudates might be an indication for the

neuroinflammation and cholinergic neuronal degeneration in the

hippocampus region. This hypothesis agrees with Sajad et al. and

Santos et al. who demonstrated a remarkable inhibition of choline

acetyl transferase, the enzyme responsible for Ach synthesis, in

addition to significant decline of SOD and CAT in an animal model

of human multiple sclerosis[49,50].

The current study demonstrated that the intra-peritoneal injection

of Spirulina exudates in mice led to remarkable disruption of the

reproductive hormones. This effect was attributed to the presence

of steroidal compounds that were identified with GC-MS and LC-

MS/MS analysis. The presence of such these compounds and their

disruptive effects on sex hormones level were correlated with the

fluctuation of monoamine neurotransmitters in the hippocampal

region of the treated animals (Figure 2). The recorded neurological

symptoms (ataxia, convulsion and comma) were attributed to the

change in the level of monoamine neurotransmitters. At the same

time, the reduction of the antioxidant enzymes activity and Ach

level in the same region might be accompanied with hippocampal

neurodegeneration due to the deteriorative effect of Spirulina

exudates treatment. Consequently, Spirulina could potentially

contribute to hazardous effects on mammalian health via producing

sterol-like compounds that might disrupt endocrine system as well

as the nervous system.

Conflict of interest statement

We declare that we have no conflicts of interest.

Acknowledgements

The authors wish to thank Prof. Dr. Refat Mohamed Ali, Prof. of

Plant Physiology, Botany Department, Faculty of Science, Fayoum

University, for his great support and valuable suggestions.

References

[1] Khan Z, Bhadouria P, Bisen PS. Nutritional and therapeutic potential of

Spirulina. Curr Pharm Biotechnol 2005; 6: 373-9.

[2] Tokusoglu O, Unal MK. Biomass nutrient profiles of three microalgae:

Spirulina platensis, Chlorella vulgaris, and Isochrisis galbana. J Food

Sci 2003; 68: 1144-8.

[3] Tang G, Suter PM. Vitamin A, nutrition, and health values of algae:

Spirulina, Chlorella and Dunaliella. J Pharm Nutr Sci 2011; 1: 111-8.

[4] Bishop WM, Zubeck HM. Evaluation of microalgae for use as

nutraceuticals and nutritional supplements. J Nutr Food Sci 2012; 2:

147-54.

[5] Cornet JF, Dubertret G. The cyanobacterium Spirulina in the

photosynthetic compartment of the MELISSA artificial ecosystem.

Marseille: Workshop on artificial ecological systems; 1990.

[6] Holman BW, Malau-Aduli AE. Spirulina as a livestock supplement and

animal feed. J Anim Physiol Anim Nutr (Berl) 2013; 97(4): 615-23.

[7] Yaakob Z, Ali E, Zainal A, Mohamad M, Takriff MS. An overview:

biomolecules from microalgae for animal feed and aquaculture. J Biol

Res 2014; 21(6): 1-10.

[8] Challouf R, Trabelsi L, Ben Dhieb R, El Abed O, Yahia A, Ghozzi K,

et al. Evaluation of cytotoxicity and biological activities in extracellular

polysaccharides released by cyanobacterium Arthrospira platensis. Braz

Arch Biol Technol 2011; 54(4): 831-8.

[9] Ananya AK, Ahmad IZ. Cyanobacteria “the blue green algae” and its

novel applications: A brief review. Int J Innov Appl Stud 2014; 7: 251-

61.

[10] Rechter S, Konig T, Auerochs S, Thulke S, Walter H, Dornenburg H,

et al. Antiviral activity of Arthrospira-derived spirulan-like substances.

Antiviral Res 2006; 72(3): 197-206.

[11] Mostafa SSM, Gawish FA. Towards to control Biomphalaria

alexandrina snails and the free living larval stages of Schistosoma

mansoni using the microalga Spirulina platensis. Aust J Basic Appl Sci

2009; 3(4): 4112-9.

[12] Lu HK, Hsieh CC, Hsu JJ, Yang YK, Chou HN. Preventive effects of

Spirulina platensis on skeletal muscle damage under exercise-induced

oxidative stress. Eur J Appl Physiol 2006; 98: 220-6.

[13] Torres-Duran PV, Ferreira-Hermosillo A, Juarez-Oropeza MA.

Antihyperlipemic and antihypertensive effects of Spirulina maxima in an

open sample of Mexican population: a preliminary report. Lipids Health

Dis 2007; 6: 33.

[14] Simpore J, Zongo F, Kabore F, Dansou D, Bere A, Nikiema JB,

et al. Nutrition rehabilitation of HIV-infected and HIV-negative

undernourished children utilizing Spirulina. Ann Nutr Metab 2005; 49:

373-80.

Samah Mamdouh Mohamed Fathy and Ashraf Mohamed Mohamed Essa/Asian Pac J Trop Biomed 2015; 5(6): 936

[15] Mao TK, Van de Water J, Gershwin ME. Effects of a Spirulina-based

dietary supplement on cytokine production from allergic rhinitis patients.

J Med Food 2005; 8: 27-30.

[16] Ravi M, De SL, Azharuddin S, Paul SFD. The beneficial effects of

Spirulina focusing on its immunomodulatory and antioxidant properties.

Nutr Diet Suppl 2010; 2: 73-83.

[17] Iwasa M, Yamamoto M, Tanaka Y, Kaito M, Adachi Y. Spirulina-

associated hepatotoxicity. Am J Gastroenterol 2002; 97: 3212-3.

[18] Jiang Y, Xie P, Chen J, Liang G. Detection of the hepatotoxic

microcystins in 36 kinds of cyanobacteria Spirulina food products in

China. Food Addit Contam Part A Chem Anal Control Expo Risk Assess

2008; 25(7): 885-94.

[19] Mazokopakis EE, Karefilakis CM, Tsartsalis AN, Milkas AN, Ganotakis

ES. Acute rhabdomyolysis caused by Spirulina (Arthrospira platensis).

Phytomedicine 2008; 15: 525-7.

[20] Banack SA, Johnson HE, Cheng R, Cox PA. Production of the

neurotoxin BMAA by a marine cyanobacterium. Mar Drugs 2007; 5:

180-96.

[21] Cox PA, Banack SA, Murch SJ, Rasmussen U, Tien G, Bidigare RR,

et al. Diverse taxa of cyanobacteria produce beta-N-methylamino-L-

alanine, a neurotoxic amino acid. Proc Natl Acad Sci U S A 2005; 102:

5074-8.

[22] Banack SA, Cox PA. Biomagnification of cycad neurotoxins in flying

foxes: implications for ALS-PDC in Guam. Neurology 2003; 61: 387-9.

[23] Esterhuizen M, Downing TG. Beta-N-methylamino-L-alanine (BMAA)

in novel South African cyanobacterial isolates. Ecotoxicol Environ Saf

2008; 71: 309-13.

[24] Papapetropoulos S. Is there a role for naturally occurring cyanobacterial

toxins in neurodegeneration? The beta-N-methylamino-L-alanine

(BMAA) paradigm. Neurochem Int 2007; 50: 998-1003.

[25] Essa AM, Fathy SM. Sex hormonal disruption by cyanobacterial

bioactive compounds. J Appl Microbiol 2014; 116: 700-9.

[26] Zarrouk C. Contribution a lˊ étude du cyanophycée. Influence de divers

facteurs physiques et chimiques sur la croissance et la photosynthése de

Spirulina maxima (Setch et Gardner) Geitl., PhD, Paris, 1966.

[27] Desikachary TV, Bai J. Taxonomic studies in Spirulina II. The

identification of Arthrospira “Spirulina” strains and natural samples of

different geographical origins. Algol Stud 1996; 83: 163-78.

[28] Clescerl LS, Greenberg AE, Eaton AD. Standard methods for the

examination of water and wastewater. 20th ed. USA: American Public

Health Association; 1999.

[29] Nishikimi N, Appaji N, Yagi K. The occurrence of superoxide anion in

the reaction of reduced phenazine methosulfate and molecular oxygen.

Biochem Biophys Res Commun 1972; 46: 849-54.

[30] Aebi H. Catalase in vitro. Methods Enzymol 1984; 105: 121-6.

[31] Lawrence RA, Parkhill LK, Burk RF. Hepatic cytosolic non selenium-

dependent glutathione peroxidase activity: its nature and the effect of

selenium deficiency. J Nutr 1978; 108(6): 981-7.

[32] Oswald C, Smits SH, Hoing M, Sohn-Bosser L, Dupont L, Le Rudulier

D, et al. Crystal structures of choline/acetylcholine substratebinding

protein ChoX from Sinorhizobium meliloti in the liganded and

unligznded-closed states. J Biol Chem 2008; 283: 32848-59.

[33] Ding XS, Li XY, Duan HY, Chung IK, Lee JA. Toxic effects of

Microcystis cell extracts on the reproductive system of male mice.

Toxicon 2006; 48: 973-9.

[34] Damkova V, Sedlackova J, Bandouchova H, Peckova L, Vitula F,

Hilscherova K, et al. Effects of cyanobacterial biomass on avian

reproduction: a Japanese quail model. Neuro Endocrinol Lett 2009; 30:

205-10.

[35] Hudson BJ, Karis IG. The lipids of the alga Spirulina. J Sci Food Agric

1974; 25: 759-63.

[36] Noaman NH, Fattah A, Khaleafa M, Zaky SH. Factors affecting

antimicrobial activity of Synechococcus leopoliensis. Microbiol Res

2004; 159: 395-402.

[37] Ramadan MF, Asker MMS, Ibrahim ZK. Functional bioactive

compounds and biological activities of Spirulina platensis lipids. Czech

J Food Sci 2008; 26(3): 211-22.

[38] Tabb MM, Blumberg B. New modes of action for endocrine-disrupting

chemicals. Mol Endocrinol 2006; 20(3): 475-82.

[39] De Coster S, van Larebeke N. Endocrine disrupting chemicals:

associated disorders and mechanisms of action. J Environ Public Health

2012; doi: 10.1155/2012/713696.

[40] Cornil CA, Dalla C, Papadopoulou-Daifoti Z, Baillien M, Balthazart

J. Estradiol rapidly activates male sexual behavior and affects brain

monoamine levels in the quail brain. Behav Brain Res 2006; 166(1):

110-23.

[41] Tucci P, Morgese MG, Colaianna M, Zotti M, Schiavone S, Cuomo V,

et al. Neurochemical consequence of steroid abuse: stanozolol-induced

monoaminergic changes. Steroids 2012; 77(3): 269-75.

[42] Takase K, Sakimoto Y, Kimura F, Mitsushima D. Developmental

trajectory of contextual learning and 24-h acetylcholine release in the

hippocampus. Sci Rep 2014; 4: 3738.

[43] Gallagher JJ, Zhang X, Hall FS, Uhl GR, Bearer EL, Jacobs RE. Altered

reward circuitry in the norepinephrine transporter knockout mouse.

PLoS One 2013; 8(3): e57597.

[44] Shiraishi M, Shibuya I, Minami K, Uezono Y, Okamoto T, Yanagihara

N, et al. A neurosteroid anesthetic, alphaxalone, inhibits nicotinic

acetylcholine receptors in cultured bovine adrenal chromaffin cells.

Anesth Analg 2002; 95(4): 900-6.

[45] Zheng P. Neuroactive steroid regulation of neurotransmitter release in

the CNS: action, mechanism and possible significance. Prog Neurobiol

2009; 89(2): 134-52.

[46] Pajovic SB, Saicic ZS. Modulation of antioxidant enzyme activities by

sexual steroid hormones. Physiol Res 2008; 57: 801-11.

[47] Pajovic SB, Saicic ZS, Spasic MB, Petrovic VM. The effect of ovarian

hormones on antioxidant enzyme activities in the brain of male rats.

Physiol Res 2003; 52: 189-94.

[48] Michos C, Kiortsis DN, Evangelou A, Karabounas S. Antioxidant

protection during the menstrual cycle: the effects of estradiol on

ascorbic-dehydroascorbic acid plasma levels and total antioxidant

plasma status in eumenorrhoic women during the menstrual cycle. Acta

Obstet Gynecol Scand 2006; 85: 960-5.

[49] Sajad M, Zargan J, Chawla R, Umar S, Sadaqat M, Khan HA.

Hippocampal neurodegeneration in experimental autoimmune

encephalomyelitis (EAE): potential role of inflammation activated

myeloperoxidase. Mol Cell Biochem 2009; 328: 183-8.

[50] Santos D, Milatovic D, Andrade V, Batoreu MC, Aschner M,

Marreilha dos Santos AP. The inhibitory effect of manganese

on acetylcholinesterase activity enhances oxidative stress and

neuroinflammation in the rat brain. Toxicology 2012; 292: 90-8.